Plant Physiology and Biochemistry 74 (2014) 33e41

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Research article

Identification and biochemical characterization of five long-chain acyl-coenzyme A synthetases from the diatom Phaeodactylum tricornutum Xiaojing Guo, Mulan Jiang, Xia Wan, Chuanjiong Hu, Yangmin Gong* Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 August 2013 Accepted 30 October 2013 Available online 7 November 2013

Long-chain acyl-CoA synthetase (ACSL; EC 6.2.1.3) catalyzes the conversion of free fatty acid to acyl-CoA ester, which is necessary for many pathways of fatty acid and lipid metabolism. The diatom Phaeodactylum tricornutum genome encodes five putative ACSLs (PtACSL1-5) that contain several highly conserved motifs and share limited sequence similarities with each other and with other known ACSLs. To verify their long-chain acyl-CoA synthetase activities, five cDNAs encoding these PtACSLs were cloned, expressed, and tested for their ability to complement the Saccharomyces cerevisiae double mutant FAA1DFAA4D. Only two of five PtACSLs were able to restore growth, facilitate exogenous fatty acid uptake, and enhance storage lipid accumulation. We also found that P. tricornutum cells are capable of importing long-chain fatty acids from extracellular environment. The identification of P. tricornutum ACSLs will provide molecular basis for the study of ACSL-mediated lipid synthesis and metabolism in diatoms. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: ACSL Fatty acids Phaeodactylum tricornutum Yeast

1. Introduction Activation of free fatty acids to their coenzyme A (CoA) thioesters is an important step necessary for the utilization of fatty acids by most lipid metabolic enzymes. Several processes such as the synthesis of phospholipids or triacylglycerols, elongation and b-oxidation of fatty acids and fatty acylation of proteins all require activated fatty acid substrates (Watkins, 1997). Thioesterification of free fatty acid to CoA is catalyzed by acyl-CoA synthetase (also known as fatty acid:CoA ligase: AMP forming; EC 6.2.1.3; ACS) by a two-step mechanism. In these ATP-dependent reactions, free fatty acid is firstly converted to an acyl-AMP intermediate through the pyrophosphorolysis of ATP. The activated carbon is then coupled to the thiol group of CoA, releasing AMP and fatty acyl-CoA (Shockey et al., 2002). Acyl CoA synthetases have been classified on the basis of the chain length of their preferred fatty acid substrates. Long-chain acyl CoA synthetase (ACSL) is the subfamily that activates fatty acids with chain lengths ranging from C12 to C20 (Soupene and Kuypers, 2008).

Abbreviations: ACSL, long-chain acyl-CoA synthetase; TAG, triacylglycerol; ACP, acyleacyl carrier protein; RACE, rapid amplification of cDNA end; QRT-PCR, quantitative reverse transcription PCR; C1-BODIPY-C12, 4,4-Difluro-5-methyl-4-bora-3,4diaza-s-indacene-3-dodecanoic acid; BSA, bovine serum albumin; VLCS/FATP, very long-chain acyl-CoA synthetase/fatty acid transport protein. * Corresponding author. Tel.: þ86 27 86838791; fax: þ86 27 86822291. E-mail address: [email protected] (Y. Gong). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.10.036

ACSL proteins share certain sequence similarity, particularly in two highly conserved amino acid motifs. One motif consists of 10e12 amino acids and is absolutely necessary for ATP binding. The presence of this motif (PROSITE PS00455) is the unifying feature of all ACS enzymes (Steinberg et al., 2000). The second motif, defined as the ACS signature motif, contains a 25-amino acid residue consensus sequence (DGWLHTGDIGXWXPXGXLKIIDRKK) that may be essential for fatty acid binding and catalytic activity of the ACS enzymes (Mashek et al., 2007). In general, eukaryotic organisms contain several isoforms of ACSL whose functions are generally not equivalent. Within a specific type of cell, multiple isoforms of ACSL may be expressed simultaneously but they can be localized to different subcellular compartments. Different subcellular localization of ACSLs could contribute to channeling fatty acids toward different anabolic and catabolic pathways (Mashek et al., 2007). For example, mitochondrial ACSLs could provide activated fatty acids for boxidation, whereas endoplasmic reticulum localized ACSLs might channel fatty acids toward neutral lipid or phospholipid synthesis. To date, our knowledge of physiological roles of ACSL has been based on the detailed genetic and biochemical studies in bacteria, yeast (Saccharomyces cerevisiae) and mammalian systems. These studies suggest that one of the most important functions of ACSL is its central role in fatty acid uptake (Mashek et al., 2007). In Escherichia coli, the single ACSL enzyme FadD and the outer membrane-bound fatty acid transport protein FadL both are required for exogenous

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long-chain fatty acid transport and activation (Black et al., 1992; DiRusso and Black, 1999). FadL acts as a receptor for fatty acids and mediates their transfer across the outer membrane. The inner membrane-associated FadD subsequently activates fatty acids concomitant with transport, resulting in the formation of acyl-CoAs. This process was defined as vectorial acylation (DiRusso and Black, 1999; Klein et al., 1971). In the yeast S. cerevisiae, four ACSLs, Faa1p, Faa2p, Faa3p, and Faa4p have been identified. Of these four ACSL isoforms, only Faa1p or Faa4 is essential for the activation of imported exogenous long-chain fatty acids with Faa1p being the major ACSL responsible for this function (Færgeman et al., 2001; Zou et al., 2003). Faa1p and/or Faa4p work in concert with a fatty acid transport protein Fat1p that is also required for fatty acid import, which thereby tightly links fatty acid uptake to ACSL-catalyzed fatty acid activation (Zou et al., 2003; Færgeman et al.,1997). In plant cells, intracellular free fatty acids are released from the triacylglycerol (TAG) molecules by lipases during seedling germination or from fatty acyleacyl carrier protein (ACP) thioesters by thioesterases localized in the plastid (Shockey et al., 2002; Schnurr et al., 2002). The model plant Arabidopsis thaliana contains nine genes encoding ACSL enzymes. Biochemical and genetic studies suggest that ACSL activities are essential for plastidial fatty acid biosynthesis and activation, cutin synthesis, production of cuticular waxes and fatty acid b-oxidation (Shockey and Browse, 2011). One field our laboratory focuses on is lipid synthesis in oleaginous microalgae. Despite the progress in microalgal lipid biosynthesis in the past decade, the knowledge of the underlying biochemistry and genetics is very limited. Given the important roles of ACSLs just described, we attempt to determine the contribution of ACSLs to lipid synthesis and metabolism in oil-rich microalgae. Reports on microalgal ACSLs are very few and thus ACSL-mediated pathways in microalgae are essentially undiscovered thus far. In the diatom Thalassiosira pseudonana, one of the eight putative ACSL genes (TplascA) was characterized and appeared to encode ACS enzyme that exhibits high activity toward long-chain polyunsaturated fatty acids (Tonon et al., 2005). In this study, we report the identification and characterization of five putative ACSL proteins from Phaeodactylum tricornutum and the data presented here provide the molecular basis for understanding the functions of ACSL enzymes in this diatom species. 2. Results 2.1. The putative P. tricornutum ACSL proteins, conserved motifs and phylogenetic analysis Diatoms are an important group of unicellular photosynthetic microalgae and are thought to contribute at least 25% of the global primary productivity in ocean ecosystems (Scala and Bowler, 2001). P. tricornutum can grow in the absence of silica and it is an attractive model species for ecological, physiological and biochemical researches in diatoms because of its short generation time, ease of culture, and successful establishment of genetic transformation and tools to generate targeted gene knockdown mutants. Although the P. tricornutum genomic sequences are available, little is known about ACSL-mediated biochemical pathways in this diatom species. Based on the annotation of the P. tricornutum genome and the results of BLAST search we identified five candidate ACSL sequences (see details in Materials and Methods 4.3). Each of the full-length coding sequences was amplified by RT-PCR and the encoded proteins were designated PtACSL1-5 (Table 1). These five putative ACSL proteins share very limited amino acid identity with each other (11e28%) and with known ACSLs from other organisms (12e41%). However, sequence analysis indicated that all PtACSLs contain a highly conserved motif that is necessary for AMP binding (Fig. 1A).

Table 1 Overview of the putative long-chain acyl-CoA synthetases identified in the genome of Phaeodactylum tricornutum. Protein name

Number of amino acids

Molecular weight

Theoretical pI

NCBI accession number

PtACSL1 PtACSL2 PtACSL3 PtACSL4 PtACSL5

721 684 702 663 678

79.0 74.7 77.2 72.7 73.5

6.81 7.55 6.04 6.91 5.93

KF359938 KF359939 KF359940 KF359941 KF359942

kDa kDa kDa kDa kDa

This AMP-binding motif was also identified in the large superfamily of acyl-activating enzymes (AAEs) that catalyze the activation of many different carboxylic acid substrates (Shockey et al., 2003). The putative ACS signature motif is also present in these 5 PtACSL proteins whereas there is an insertion of 1, 5, and 9 amino acid residue(s) in this motif of PtACSL2, PtACSL3 and PtACSL4, respectively, when compared with that of E. coli and yeast ACSL homologs (Fig. 1B). Insertion of these amino acid residues in this region might indicate their distinct enzymatic activities or functions. As the ACSL-specific sequence determinant, a linker domain was also observed in these 5 candidate PtACSLs. This linker domain is specifically present in almost all eukaryotic ACSLs but absent in FadD, the sole ACSL enzyme in E. coli, and other members of the AMPbinding protein family such as the 4-coumarate-CoA ligases (Fig. 1C) that also contain the AMP-binding signature motif (Shockey et al., 2002). Of 5 PtACSLs, PtACSL2 contains the longest linker domain with an extension of 70 amino acid residues. In addition to these conserved domains, an invariant proline residue located 24-30 amino acids downstream of AMP-binding domain and an invariant histidine residue followed by the proline residue are also remarkably conserved in all ACSL proteins (Fig. S1). Other conserved regions include five invariant amino acid residues located upstream and downstream of ACS signature motif. These highly conserved motifs and amino acid residues may be essential for the activity of ACSL enzymes. The hydrophobicity plot predicted one transmembrane region at the N-terminus of PtACSL1 (Fig. S2), whereas no probable transmembrane sequence was detected in other P. tricornutum ACSL proteins. To examine the phylogeny of PtACSLs and other known ACSL proteins, we performed neighbor-joining analysis based on the full amino acid sequences of ACSL and several proteins from very longchain acyl-CoA synthetase/fatty acid transport protein (VLCS/FATP) family. The members of VLCS/FATP protein family also possess AMP-binding domain but show specificity for fatty acid substrates of very long chain length (22) (Steinberg et al., 2000). Phylogenetic analysis indicates that ACSL proteins from P. tricornutum, plant, animal and fungi are distinctly separated from FATP/VLACS proteins (Fig. 1D). Of 5 PtACSLs, PtACSL5 is clustered with animal ACSL proteins and evolutionary relationship of PtACSL1 may be unresolved due to lack of a strong bootstrap support, while each of other 4 PtACSLs forms an independent cluster outside several clades of the ACSL protein family (Fig. 1D). These analyses may suggest several distinct origins for PtACSLs with divergent functions during their evolution and thus the biological roles of each of these PtACSL proteins may be unique.

2.2. Fatty acid transport is restored upon expression of PtACSL1 or PtACSL4 in the yeast FAA1DFAA4D double disrupted strain To determine which of the cloned cDNAs of PtACSLs indeed encode active enzymes that are able to activate long-chain fatty acids, the coding sequences of 5 PtACSLs were cloned into the

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Fig. 1. Sequence analysis of deduced amino acids of P. tricornutum ACSL homologs. (A) Amino acid sequence alignment of the AMP-binding domain signature motif from five PtACSL homologs. (B) Consensus sequences of the ACS signature motif from PtACSLs and ACSL orthologues from E. coli and yeast (C) Linker domain specifically present in ACSL proteins. (D) Phylogenetic relationships among deduced amino acid sequences of ACSL proteins and the VLCS/FATP1 family proteins (shaded in gray). The tree was constructed according to the Neighbor-Joining algorithm. GenBank accession numbers are given in Table S1. Numbers at branch points are bootstrap percentages derived from 1000 replicates.

yeast expression vector pYES2/CT downstream of the galactose inducible promoter. These constructs as well as the empty vector were transformed individually into a S. cerevisiae strain YB525 and allowed for expression and complementation analysis. The yeast strain YB525 lacks both FAA1 and FAA4 that encode ACSL proteins Faa1p and Faa4p, respectively, together accounting for over 90% of the long-chain acyl-CoA synthetase activity. In S. cerevisiae, both fatty acid transport protein Fat1p and Faa1p/ Faa4p are essential for import and activation of exogenous fatty acids, which is especially important for maintaining the viability of yeast cells when cellular fatty acid synthase (Fas) is inactived by a specific inhibitor (cerulenin). Yeast cells thus become auxotrophic for long-chain fatty acids when grown on media containing cerulenin. Wild type strain can grow on media containing long-chain fatty acids and cerulenin, whereas YB525 with deletions of both FAA1 and FAA4 is not viable under the same condition due to functional loss of fatty acid transport. Activation of exogenous fatty acids by ACSL is thought to be specifically coupled with Fat1p-mediated fatty acid transport and lack of ACSL activity leads to a fatty acid transport defect (Færgeman et al., 2001; Zou et al., 2003). Since the activity of one ACSL enzyme is sufficient to restore growth of YB525 under the synthetic lethal condition as just described, we attempted to determine whether the growth defect of YB525 could be alleviated or abolished following expression of one of PtACSLs. Expression of each of PtACSLs tagged with a C-terminal V5 epitope was verified by Western blot following induction with galactose. The amount of each recombinant PtACSL was detectable to varying extents (Fig. 2A). Many attempts to improve protein yield of PtACSL2 in YB525 were made but its yield was still low. These strains were then used for all further studies. Growth of these strains was firstly evaluated in a plate assay. As shown in Fig. 2B, YB525 derivative strains carrying empty vector or PtACSL showed a growth defect on media containing only

45 mM cerulenin. Addition of 100 mM oleate to the media did not alleviate the defective growth phenotype of the YB525 strain carrying empty vector, whereas growth of YB525 strains carrying PtACSL1 or PtACSL4 was restored under the same condition. The mutant phenotype was successfully complemented by PtACSL1 or PtACSL4, indicating that both of them expressed an active ACSL enzyme and could interact with the components of yeast fatty acid transport system. Other 3 isoforms of PtACSL did not complement the mutant phenotype, suggesting that other factors such as appropriate subcellular targeting and physiological function played by the ACSL enzyme also contribute to phenotypic restoration in the mutant YB525. Another phenotype of YB525 strain is that fatty acid import is restricted due to a defective functional link between Fat1pmediated fatty acid uptake and Faa1p/Faa4p-mediated fatty acid activation. It was demonstrated that Faa1p/Faa4p was also required for fatty acid transport process besides Fat1p (Zou et al., 2003). To determine whether expression of each of PtACSLs can restore the ability of fatty acid import in yeast, we used confocal laser scanning microscope to monitor the intracellular accumulation of the fluorescent long-chain fatty acid analog C1-BODIPYC12 in wild-type S. cerevisiae strain and the YB525 strains expressing each of the PtACSL genes or carrying empty vector. Wild-type yeast cells accumulate large amounts of C1-BODIPY-C12 quickly, whereas accumulation of C1-BODIPY-C12 was considerably reduced in the YB525 strain carrying empty vector (Fig. 3). Previous analysis of fatty acid import of mutants with deletion of different ACSL genes suggested that of the four ACSL isoforms, Faa1p is the predominant ACSL enzyme required for fatty acid uptake by a vectorial acylation process in S. cerevisiae (Færgeman et al., 2001; Zou et al., 2003). In our study, expression of PtACSL1 or PtACSL4 restored the accumulation of C1-BODIPY-C12 into cells of YB525 strain, but other 3 PtACSLs did not. These results support that PtACSL1 or PtACSL4 could function with yeast transport

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A 105 kDa 71 kDa 50 kDa

B 100 10-1 10-2 10-3 10-4 100 10-1 10-2 10-3 10-4 100 10-1 10-2 10-3 10-4 pYES2/CT PtACSL1 PtACSL2 PtACSL3

PtACSL4 PtACSL5 YNBGR-Cer

YNBGR-Ole

YNBGR-Cer-Ole

Fig. 2. PtACSL1 and PtACSL4 complement growth defect of YB525 under fatty acid auxotrophic conditions. (A) Expression of P. tricornutum ACSL isoforms was verified by immunoblotting with an anti-V5 antibody. Each lane contained 5 mg total proteins extracted from yeast cells. Lane 1, protein ladder; Lane 2, pYES2/CT; Lane 3-7, PtACSL1-5. (B) The growth defect of YB525 cells under fatty acid auxotrophic conditions is rescued by PtACSL1 or PtACSL4. YB525 cells harboring empty vector or expressing the relevant P. tricornutum ACSL isoform as indicated were serially diluted tenfold and spotted onto YNBGR media containing both oleate (Ole) and cerulenin (Cer), oleate only and cerulenin only.

protein Fat1p in fatty acid uptake in a manner analogous to that of Faa1p. In the complementation experiment described above, growth of YB525 strains expressing PtACSL1 or PtACSL4 under fatty acid auxotrophic condition correlated well with the restored abilities of import and subsequent metabolic utilization of exogenous fatty acids. We also used confocal laser scanning microscope to monitor accumulation of C1-BODIPY-C12 in P. tricornutum. We observed that P. tricornutum cells accumulate exogenous C1-BODIPY-C12 quickly (within 3e5 min), whereas no C1-BODIPY-C12 accumulation was observed when cells were grown in the absence of the fluorescent analog (Fig. 4). This observation indicates that ACSL enzymes from

Wild type

PtACSL2

PtACSL3

P. tricornutum might play certain roles in activation and utilization exogenous fatty acids from the environment. 2.3. Lipid body accumulation is enhanced upon expression of PtACSL1 or PtACSL4 Because ACSL-mediated reaction may also be involved in several metabolic pathways such as complex lipid synthesis, we wanted to address whether PtACSL1 or PtACSL4 was associated with this process in YB525 strain. In yeast, TAGs and other neutral lipids accumulate within the organelle known as lipid bodies. Acyl-CoAdependent esterification reactions make important contribution

pYES2/CT

PtACSL4

PtACSL1

PtACSL5

Fig. 3. Confocal laser scanning microscope of wild type strain and the YB525 derivative strains following incubation with the fluorescent long-chain fatty acid analogue C1BODIPY-C12.

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Control

37

+ C1-BODIPY-C12

Fig. 4. Fatty acid import by P. tricornutum cells. Confocal laser scanning microscope was used to monitor the accumulation of fluorescent long-chain fatty acid C1-BODIPY-C12. Cells not incubated with C1-BODIPY-C12 were used as control.

to the synthesis of these storage lipids (Sandager et al., 2002), in which the ACSL enzymatic activities are required for activation of free fatty acids as cosubstrates. In addition, Faa1p and Faa4p are the components of lipid particles (Athenstaedt et al., 1999). We stained yeast cells using the fluorescent dye Nile Red to visualize lipid bodies. As shown in Fig. 5, the strain YB525 lacking both FAA1 and FAA4 showed significantly reduced lipid bodies compared with that of wild type, whereas expression of PtACSL1 or PtACSL4 enhanced lipid body accumulation in YB525 due to increased synthesis of TAGs and other storage lipids. Expression of other 3 PtACSL had no impact on lipid body accumulation in the mutant YB525. These data support a role of PtACSL1 or PtACSL4 in channeling newly synthesized fatty acids to the storage site of neutral lipids, the lipid body.

(day 0) after 2 days of nitrogen starvation, which was followed by a decrease during the next 3 days. A slight increase (1.5-fold) was observed in the expression level of PtACSL5 after 1 day of nitrogen starvation and it decreased to the minimum in the following days. Nitrogen starvation resulted in a similar down-regulation of PtACSL2, PtACSL3, and PtACSL4, while PtACSL3 expression was slightly increased after day 3 during nitrogen starvation. These PtACSL isoforms do not seem to be associated with storage lipid synthesis under nitrogen starvation condition. Alternatively, the biochemical reactions catalyzed by these PtACSL enzymes might not be the committing step of storage lipid synthesis under this condition.

2.4. Expression patterns of PtACSLs under nitrogen starvation

Long-chain acyl-CoA synthetase belongs to the acyl-activating enzyme superfamily, which contains a highly conserved signature sequence (PROSITE entry PS00455) necessary for AMP binding and adenylate formation. Search of the well annotated P. tricornutum genome database and analysis of signature sequence enable us to identify 5 putative ACSL proteins. Besides

We examined the expression profiles of each of the PtACSL genes in response to nitrogen starvation by quantitative reverse transcription PCR (QRT-PCR). As shown in Fig. 6, the expression of PtACSL1 mRNA increased to a maximum of 4-fold above control

Wild type

PtACSL2

3. Discussion

pYES2/CT

PtACSL3

PtACSL1

PtACSL4

PtACSL5

Fig. 5. Nile Red staining showing neutral lipid accumulation in wild type strain and the YB525 derivative strains harboring empty vector or expressing the relevant P. tricornutum ACSL isoform as indicated.

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Fig. 6. Expression pattern for PtACSL gene transcripts under nitrogen starvation. QRT-PCR was performed with primers for amplification of each of PtACSL transcripts. The transcript abundance was normalized to that of housekeeping gene PtTBP (TATA box binding protein).

the AMP-binding domain, the ACS signature motif and ACSLspecific linker domain are also present in these 5 PtACSL proteins, which thus represent the entire ACSL protein family in P. tricornutum. In order to verify their acyl-CoA synthetase activities, a genetic complementation strategy in yeast was attempted. The full-length coding sequences for each of candidate PtACSLs were expressed individually in the yeast mutant YB525, which lacks both FAA1 and FAA4 and thus exhibits growth defect under fatty-acid auxotrophic condition and displays fatty-acid-uptake defect. We found that of 5 PtACSLs, only PtACSL1 and PtACSL4 could restore the growth phenotype and the ability of fatty acid uptake. Expression of these 2 PtACSL isoforms also enhanced intracellular accumulation of neutral lipids, suggesting their role in fatty acid trafficking. Because Faa1p was thought to be the principle ACSL for coupling of fatty acid import and utilization, Faa1p appears to be functionally replaced, at least partially, by P. tricornutum ACSL ortholog PtACSL1 or PtACSL4. The yeast double mutant (FAA1DFAA4D) is a well-characterized model system to identify candidate ACSL genes. In the past decade, a number of ACSL genes have been identified by researcher in different fields. Their results showed that growth defect of the double mutant under fatty-acid auxotrophic condition could be rescued by plant orthologues (Arabidopsis LACS1-5, LACS8-9) (Shockey et al., 2002) and animal orthologues (rat ACSL1, 4, and 6) (Tong et al., 2006). The functional conservation of Faa1p in fatty acid uptake might thus be associated with those highly conserved domains and amino acid residues identified in this study (Fig. 1A and B; Fig. S1). Among the Arabidopsis ACSL protein family, both LACS6 and LACS7 are localized to peroxisome and thus were unable to complement the yeast double mutant (Fulda et al., 2002). While the roles of fatty acid uptake and trafficking in other 7 Arabidopsis LACSs were not characterized, rat ACSL1, 4, and 6, like P. tricornutum ACSL1 and 4, were able to facilitate fatty acid uptake and enhance storage lipid accumulation (Tong et al., 2006). Because fatty acid uptake and trafficking of endogenously synthesized fatty acids to intracellular lipid bodies are two independent processes, these functional ACSL enzymes appear to be able to provide long-chain acyl-CoA synthetase activities for distinct pathways. Of 5 PtACSLs, PtACSL5 was predicted to have a potential peroxisomal localization, which not only might be responsible for its inability to complement growth phenotype of the mutant, but also suggests its possible involving in fatty acid b-oxidation in P. tricornutum.

P. tricornutum is a unicellular microorganism. The presence of multiple ACSL proteins raises the question of whether P. tricornutum cells, like E. coli and yeast, are able to import fatty acids from the environment. To answer it, we incubated P. tricornutum cells with the fluorescent fatty acid analog C1-BODIPY-C12 and observed the accumulation of C1-BODIPY-C12 within P. tricornutum cells. This observation suggests the ability of P. tricornutum cells in fatty acid uptake, which might be important for P. tricornutum cells when they are grown under carbon limitation conditions. It was reported that free fatty acids are present in low concentrations in marine environment (Parrish, 1988), which may be caused by the degradation of lipid compounds. These available free fatty acids may be taken up and utilized by some phytoplankton species such as diatoms. If fatty acid uptake takes place in P. tricornutum cells by vectorial acylation similar to that in yeast, the components of fatty acid uptake await further identification. In photosynthetic microalgae, de novo fatty acid synthesis occurs primarily in the chroloplast with ACP as acyl acceptor. When the fatty acid reaches the appropriate length, an acyl-ACP thioesterase can hydrolyze the acyl ACP and release free fatty acid, which is then targeted for export from the chloroplast and reactivated as acyl-CoAs (Hu et al., 2008; Radakovits et al., 2010). As in higher plants, one or more plastidial ACSLs from P. tricornutum may be essential for reactivation of these free fatty acids. It must be noted that unlike plant plastids, diatom plastids are surrounded by four membranes (Gruber et al., 2007), suggesting that fatty acid export from the plastids in diatom might be distinct from that in plants. However, analysis of amino acid sequences of P. tricornutum ACSLs revealed a lack of the conserved motif (ASAFAP) that is particularly important for protein plastid targeting in diatom. Further subcellular localization, genetic analysis and characterization of substrate specificity of all PtACSLs will provide insight into the specific roles of these enzymes in lipid synthesis and other biological processes. 4. Materials and Methods 4.1. Algal strain and culture condition The diatom P. tricornutum Pt9 (CCMP633) was kindly provided by Dr. Hanhua Hu of Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). P. tricornutum was grown in f/2-enriched artificial sea water (f/2AW, pH8.5) medium (Harrison et al., 1980) bubbled with a sterile air at a temperature of 25  C under

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continuous illumination (50 mmol photons m2 s1) provided by daylight fluorescent tubes. For gene expression analysis under nitrogen starvation, cells grown in nitrogen-replete medium (882 mM NaNO3) at mid-log phase were centrifuged (2000 g for 5 min), washed twice, and resuspended in nitrogen-free f/2 medium for further culture. Cultures maintained under nitrogen starvation condition for 1d, 2d, 3d, 4d, and 5d were harvested and the cell pellets were frozen in liquid nitrogen before storage at 70  C. These samples were used for RNA extraction and further quantitative reverse transcription PCR (QRT-PCR) analysis. 4.2. Yeast strains, media and culture conditions Yeast extract, peptone, yeast nitrogen base and agar were obtained from Difco. Oleate and cerulenin were obtained from Sigma. All other chemicals were obtained from standard suppliers. The S. cerevisiae strains used in this study are listed in Table 2. For functional expression of specific proteins under the control of the GAL1 promoter, the expression constructs were firstly transformed into chemically competent YB525 cells according to the PEG/ lithium acetate method (Elble, 1992). Transformants were selected by plating on synthetic minimal defined media (SC) containing 0.67% yeast nitrogen base (YNB) with ammonium sulfate, 2% (w/v) raffinose, 100 mg/L adenine, amino acids as required (arginine,

Table 2 Microbial strains and primers used in this study. Strain or primers Strains E. coli TransT1 Wild type (S. cerevisiae) YB525 (S. cerevisiae)

YB525:pYES2/CT YB525:PtACSL1 YB525:PtACSL2 YB525:PtACSL3 YB525:PtACSL4 YB525:PtACSL5 Primers PtACSL1-For PtACSL1-Rev PtACSL2-For PtACSL2-Rev PtACSL3-For PtACSL3-Rev PtACSL4-For PtACSL4-Rev PtACSL5-For PtACSL5-Rev 5UPM-1 5NUP-1 PtACSL2R563-1 PtACSL2R563-2 PtACSL2R563-3 Q-PtACSL1-For Q-PtACSL1-Rev Q-PtACSL2-For Q-PtACSL2-Rev Q-PtACSL3-For Q-PtACSL3-Rev Q-PtACSL4-For Q-PtACSL4-Rev Q-PtACSL5-For Q-PtACSL5-Rev

Use, relevant characteristic (s), and/or sequence (source) E. coli host for DNA manipulations, TransGen (Beijing, China) MATa ura3-52 his3D200 ade2-101 lys2-801 leu2-3, 112 MATa ura3-52 his3D200 ade2-101 lys2-801 leu2-3, 112 faa1D::HIS3 faa4D:LYS2, kindly provided by Dr. Kehou Pan, Ocean University of China YB525 mutant harboring the yeast expression vector pYES2/CT YB525 mutant harboring PtACSL1 YB525 mutant harboring PtACSL2 YB525 mutant harboring PtACSL3 YB525 mutant harboring PtACSL4 YB525 mutant harboring PtACSL5 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50

GGGGTACCATGAAATACCTTTTGTTCTTGC 30 GCTCTAGAGAAGATGCCTTTCTTTTTTAC 30 CCCGGATCCATGCGTGATACCCTACGAAA 30 CCCTCGAGCTTCGACAACTCGTCCTCA 30 CCCAAGCTTATGACATCGTTGGCTACGAC 30 CCGCTCGAGGGCTGCTTTGCGCTGTAC 30 GGGGTACCATGAGCTCGTTGCCTCTTTC 30 GCTCTAGACGCGTACATGGCGTCGAT 30 GGGGTACCATGACGAACCAAAATATGCG 30 GCTCTAGAAAGCTTGCTGAGCGGAGG 30 CTAATACGACTCACTATAGGGC 30 AAGCAGTGGTATCAACGCAGAGT 30 GCCGCACTTTTCCGTA 30 CATGTGTTCATCGAGGAGAGC 30 CAGAGCTGGGGACAATTTCGT 30 CTGGCGCATATTTTGGAACT 30 ACTGTTCCAAGGCACCAATC 30 CGACCCAAAGGTGTTATGCT 30 GATATGCCAGACCGGAAGAA 30 ATGTTTCATACGGGAGATTTGG3’ ATTGGCGTAGGACAAACGTACT 30 GAAGGAATATCGTCAGCAGGTC 30 GGGAGCGTTGAATCCAATAATA 30 AAGTGGAACAACTGGCAACC 30 AAATATGGGCCAAAGGAAGG 30

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cysteine, leucine, lysine, threonine, and tryptophan (100 mg L1); aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, and valine (50 mg L1)), and agar (2%). Cultures were grown at 30  C for 2e3 days. Representative positive colonies were chosen at random and grown in SC medium at 30  C until mid-log phase. The expression of PtACSL genes from the GAL1 promoter was induced by supplementing galactose to 2% (w/v). For yeast mutant complementation, cells of the YB525 derivative strains were grown for 8e12 h under induction condition, harvested, washed with SC medium and resuspended to an optical density (OD) of 0.4 in SC medium. Samples were serially diluted in SC medium (101e104) and 2 mL of each dilution was spotted to three different YNBGR plates (YNBGR-Ole, YNBGR plate supplemented with 100 mM oleic acid; YNBGR-Cer, YNBGR plate supplemented with 45 mM cerulenin; YNBGR-Ole-Cer, YNBGR plate supplemented with 100 mM oleic acid and 45 mM cerulenin). The plates were incubated at 30  C for 3e4 d. 4.3. Search and identification of the candidate long-chain acyl-CoA synthetases from P. tricornutum The candidate ACSL proteins were screened from the annotated genome database of P. tricornutum (http://genome.jgi-psf.org/ Phatr2/Phatr2.home.html) by BLAST using amino acid sequences of the ACSL proteins from plant, animal, and the diatom T. pseudonana as queries, and by searching of the annotated domain information using PROSITE PS00455 (AMP-binding signature motif) as key word. The screened sequences were further analyzed to confirm that candidate sequences contained the eukaryotic ACSLspecific linker domain. This linker domain is generally located between two conserved domains, LS1 and LS2, which were previously identified when two rat (Rattus norvegicus) ACSL protein sequences and that of the clickbeetle (Pyrearinus termitillumanans) luciferase were compared (Fujino and Yamamoto, 1992). The linker domain was specifically found in many eukaryotic ACSL proteins, but not found in E. coli ACSL and other eukaryotic acyl-CoA synthetases that utilize short-chain, medium-chain or very long-chain fatty acid as their preferred substrates. 4.4. Cloning of coding sequences of the candidate PtACSLs To clone the coding sequence of each of putative PtACSL proteins, RT-PCR and rapid amplification of cDNA end (RACE) were conducted. Based on the available sequence information of genome database, we found that of these 5 putative PtACSLs, only PtACSL2 lacks the full-length coding sequence. To determine the transcriptional start point of PtACSL2, we carried out 50 RACE. Total RNA was extracted from P. tricornutum cells grown at mid-log phase using TRIzol Reagent (Takara) according to the manufacturer’s instruction. Genomic DNA was removed from total RNA with gDNA Eraser (Takara) at 37  C for 15 min. Gene specific primers were designed according to the sequence information from the P. tricornutum genome database. cDNA tailed with SMART II A oligonucleotide sequence was synthesized using a SMART RACE cDNA amplification kit (Clontech, Japan) and the reverse primer PtACSL2R563-1. The 50 end cDNA fragments were generated by nested PCR. The first PCR was performed using cDNAs as template, and the second PCR using a 1:50-diluted product of the first PCR as the template. The primer pair PtACSL2R563-2 and 5UPM-1 were used in the first PCR, and the primer pair PtACSL2R563-3 and 5NUP-1 were used in the nested PCR. The primers are listed in Table 2. The PCR products were cloned into pEASYTM-T1 Simple vector and sequenced. For RT-PCR amplification of full-length coding sequence of each of PtACSLs, RNA extraction and genomic DNA digestion were performed as described above. Total cDNAs were synthesized from

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digested RNAs with an oligo(dT) primer and reverse transcriptase using the PrimeScriptÒ RT reagent Kit (Takara). Two mL of the resulting cDNA was used for amplification of coding sequences of each putative PtACSL with gene specific primers containing yeast consensus sequence for enhanced translation adjacent to the start codon. The sequences of all oligonucleotide primers used in this study are listed in Table 2. The amplified PCR products were cloned into pEASYTM-T1 Simple vector using pEASY-T1 Simple Cloning Kit (TransGen Biotech., China) and the corresponding clones were verified by DNA sequencing. 4.5. Sequence analysis Database similarity searches were conducted against the Phaeodactylum tricornutum genome database (http://genome.jgipsf.org/Phatr2/Phatr2.home.html) using BLASTN and BLASTX programs. Sequence comparisons were made using Lasergene software (DNAStar, Madison, WI, USA). Transmembrane regions were predicted by the TMHMM Server (http://www.cbs.dtu.dk/services/ TMHMM-2.0/). Prediction of peroxisomal target signals was conducted using the PTSs predictor (http://www.peroxisomedb.org/ Target_signal.php). Prediction of open reading frame (ORF) of PtACSL2 coding sequence was made by the EditSeq program (version 3.88). The theoretical molecular weight and isoelectric point (pI) of the deduced polypeptides were computed by the Compute pI/Mw server (http://web.expasy.org/compute_pi/). Protein sequence alignments were conducted using the ClustalX program or the ESPript server (version 2.2) (http://espript.ibcp.fr/ ESPript/cgi-bin/ESPript.cgi) and phylogenetic tree was constructed from the alignments using MEGA 4.0 using the amino acid sequences of ACSL proteins described in Table S1. 4.6. Plasmid construction Molecular manipulations were performed by standard protocols. Molecular tool enzymes were used according to instructions provided by the manufacturers. Primers are listed in Table 2 and plasmids are listed in Table S2. For complementation of the S. cerevisiae mutant YB525 (FAA1DFAA4D), each full-length coding sequence of P. tricornutum ACSL was PCR amplified using gene specific primer pairs with total cDNAs from P. tricornutum cells as template. The accuracy of cloned PCR fragments was confirmed by sequencing. (i) The plasmid used to express PtACSL1. The stop codonremoved coding sequence of PtACSL1 was generated by PCR using prime pair PtACSL1-for/rev and cloned into pEASY-T1 Simple, resulting in pOCGXJ17. The PtACSL1 was excised from pOCGXJ17 with KpnI plus XbaI and ligated into the same sites of pYES2/CT (the S. cerevisiae expression vector) to generate pOCGXJ4. (ii) The plasmid used to express PtACSL2. The stop codonremoved coding sequence of PtACSL2 was generated by PCR using prime pair PtACSL2-for/rev and cloned into pEASY-T1 Simple, resulting in pOCGXJ23. The PtACSL2 was excised from pOCGXJ23 with BamHI plus XhoI and ligated into the same sites of pYES2/CT to generate pOCGXJ24. (iii) The plasmid used to express PtACSL3. The stop codonremoved coding sequence of PtACSL3 was generated by PCR using prime pair PtACSL3-for/rev, which introduced a HindIII and XhoI restriction site, respectively. The resulting PCR product was digested with HindIII plus XhoI and cloned into the HindIII-XhoI sites of pYES2/CT to generate pOCGXJ9.

(iv) The plasmid used to express PtACSL4. The stop codonremoved coding sequence of PtACSL4 was generated by PCR using prime pair PtACSL4-for/rev and cloned into pEASY-T1 Simple, resulting in pOCGXJ11. The PtACSL4 was excised from pOCGXJ11 with KpnI plus XbaI and ligated into the same sites of pYES2/CT to generate pOCGXJ22. (v) The plasmid used to express PtACSL5. The stop codonremoved coding sequence of PtACSL5 was generated by PCR using prime pair PtACSL5-for/rev, which introduced a KpnI and XbaI restriction site, respectively. The resulting PCR product was digested with KpnI plus XbaI and cloned into the KpnI-XbaI sites of pYES2/CT to generate pOCGXJ10.

4.7. BODIPY-labeled fatty acid uptake Assessment of fatty acid import capacity was performed as described by Li et al. (Li et al., 2005). Yeast cells (vector/FAA1DFAA4D and PtACSL/FAA1DFAA4D) were cultured in SC media containing glucose at 30  C for 24 h followed by 8e10 h of growth in galactosecontaining SC media to induce gene expression. Cells of P. tricornutum were grown in f/2 medium to mid-log phase. Cells (yeast or P. tricornutum) were harvested by centrifugation and resuspended in phosphate-buffered saline (PBS) at a cell density of 6  107/mL before use. All steps were performed at room temperature. The fluorescent long-chain fatty acid analog 4, 4-difluro-5-methyl-4bora-3, 4-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12) was prepared in PBS buffer containing 15 mM fatty acid-free bovine serum albumin (BSA). The reaction was initiated by adding 50 mL of cells in a 1.5-ml microcentrifuge tube and incubated with equal volume of 5 mM C1-BODIPY-C12 in the dark for 3e5 min. Before fluorescent observation, 50 mL of trypan blue (final concentration 0.33 mM) was added to the reaction to reduce background fluorescence. Fatty acid import was assessed using a confocal laser scanning microscopy to detect accumulation of the fluorescent long-chain fatty acid analogue C1-BODIPY-C12 in these labeled cells. Cells were visualized on a Nikon confocal laser scanning microscopy (Nikon A1R-si) equipped with a FITC filter and a 200 objective. An argon laser source was used for imaging with excitation at 488 nm and emission at 505 nm. For fluorescence imaging of yeast cells, the instrument settings for pinhole, detection gain, amplification offset, and amplification gain were optimized for cells of the YB525 strain harboring empty vector to ensure that the microscope was set for its full dynamic range. For fluorescence imaging of P. tricornutum cells, the instrument settings were optimized for wild-type P. tricornutum cells without treatment of fluorescent long-chain fatty acid analogue C1-BODIPY-C12. The same settings were used for all subsequent image collections. 4.8. Nile Red staining Yeast cells were grown to mid-log phase under inducing conditions as described above and incubated with 1 mg/mL Nile red (stock 0.5 mg/mL dimethylsulfoxide) in the dark for 10 min. The cells were subsequently washed with H2O twice, and immediately observed by a confocal scanning laser microscopy (Nikon A1R-si). 4.9. Western blotting Proteins (5 mg) from yeast cells were extracted using the method of post-alkaline extraction as described by Kushnirov (Kushnirov, 2000), separated by SDS-polyacrylamide gel electrophoresis (SDSPAGE) and transferred onto a nitrocellulose membrane (Millipore). The membrane was incubated with the anti-V5 mouse antibodies (Invitrogen) at 1:5000 dilution and alkaline phosphatase-

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conjugated goat-anti-mouse secondary antibodies (Roche) at 1:1000 dilution. Alkaline phosphatase activity was visualized with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates (Amresco). 4.10. Quantitative reverse transcription-PCR (QRT-PCR) Total RNA extraction and genomic DNA digestion were performed as described in 4.4. cDNA was synthesized from digested RNAs using the PrimeScriptÒ RT reagent Kit (Takara). Two mL of the resulting cDNA was used for QRT-PCR with the SYBR Green I master mix Kit (Takara). Reactions were run in a MiniOpticon Thermal Cycler (Bio-Rad). Each PtACSL cDNA was amplified with primer pairs listed in Table 2. The constitutively expressed housekeeping gene TBP (encoding TATA box binding protein) was used as an internal standard. Gene expression analyses were performed with the method of 2DDCt (Livak & Schmittgen, 2001). Acknowledgments We thank Dr. Kehou Pan of Ocean University of China for providing the S. cerevisiae ACSL-deficient double mutant YB525, and Dr. Hanhua Hu of Institute of Hydrobiology, Chinese Academy of Sciences for providing the P. tricornutum strain. This study was supported by National Natural Science Foundation of China Grants 31100072, 31270346 and National Hi-Tech Program of China Grant 2011AA100904. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2013.10.036. References Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S.D., Daum, G., 1999. Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae. J. Bacteriol. 181, 6441e6448. Black, P.N., DiRusso, C.C., Metzger, A.K., Heimert, T.L., 1992. Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme synthetase. J. Biol. Chem. 267, 25513e25520. DiRusso, C.C., Black, P.N., 1999. Long-chain fatty acid transport in bacteria and yeast. Paradigms for defining the mechanism underlying this protein-mediated process. Mol. Cell Biochem. 192, 41e52. Elble, R., 1992. A simple and efficient procedure for transformation of yeasts. BioTechniques 13, 18e20. Færgeman, N.J., DiRusso, C.C., Elberger, A., Knudsen, J., Black, P.N., 1997. Disruption of the Saccharomyces cerevisiae homologue to the murine fatty acid transport protein impairs uptake and growth on long-chain fatty acids. J. Biol. Chem. 272, 8531e8538. Færgeman, N.J., Black, P.N., Zhao, X.D., Knudsen, J., DiRusso, C.C., 2001. The acyl-CoA synthetases encoded within Faa1 and Faa4 in Saccharomyces cerevisiae function

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